1. Field
The present disclosure relates to a method of sequential and multiple immunostaining for detection of various antigens in the same specimens.
2. Description of the Related Art
In general, cancer diagnosis mainly relies on diagnostic methods based on histopathology. However, these methods have disadvantages that they cannot guarantee to identify any cancer cells present in tissues from which specimens were collected, and also have limitations in accurately identifying the metastasis of a given cancer to another part of a body. Recently, with improved accuracy in cytoscopy a more convenient and accurate diagnosis has been available. Cancer diagnosis may be performed using cells extracted from tissues or those separated from a body fluid such as blood and lymph. However, their lesions are relatively small in size and have a limited number of cells thus limiting the number of tests.
Circulating tumor cells (CTCs) are a type of tumor cells present in a very small amount in the blood of a metastatic cancer patient. CTCs may be discovered from a patient before any tumor is initially detected from the patient. In some cases, CTCs may be also found in cancer patients even after cancer cells have been removed by surgery. Detection, separation, and analysis of separated CTCs are very helpful in early diagnosis of cancers, early diagnosis of cancer metastasis, and prediction of cancer recurrence. However, it is very difficult to accurately analyze the characteristics of the CTCs because they are present only in trace amount and also the cells are very fragile.
In order to detect the presence of cancer cells in a solution, such as blood where various cells with various characteristics are mixed therein, it is necessary to use at least three different kinds of dyes including Cytokeratin, CD45, and nuclear stain. When cells are subject to immunostaining using a dye for color formation or a fluorescent dye there is a limitation in the number of dyes to be used simultaneously. Therefore, there are only a limited number of dyes which may be used for the analysis of genes and proteins of the identified cancer cells. In the related art, when multiple antigen-antibody reactions are performed on the same specimens it usually entails a process of separating antibodies from the antigens. This process may cause temporary modification of protein structure at a condition of very low pH or adjusted ionic concentration, which will damage cells and thereby affect the result of the subsequent tests.
Various tests may be performed using a limited number of specimens in studying the characteristics of cancer cells, providing therapies, selecting therapeutic drugs, and determining prognosis. Therefore, there is a need for the development of a method for testing various antigens using a limited number of specimens without damaging their cells.